Generation of multiply charged peptides and proteins from glycerol-based matrices using lasers with ultraviolet, visible and near-infrared wavelengths and an atmospheric pressure ion source

Abstract

Conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is characterized by the predominant generation of singly charged analyte ions. Recent studies demonstrated that high charge states of peptides and small proteins can be produced with an atmospheric pressure (AP) MALDI ion source if liquid glycerol-based matrix systems are utilized and the AP-vacuum transfer is realized via a heated ion inlet tube [R. Cramer, A. Pirkl, F. Hillenkamp, K. Dreisewerd, Angew. Chem. Int. Ed. 52 (2013) 2364–2367]. Here, we used this AP ion source and employed an optical parametric oscillator (OPO) laser to study the wavelength dependence of the ion generation between 260 and 1080 nm. Three matrix systems with different optical absorption characteristics were investigated: non-absorbing glycerol mixed with trifluoroacetic acid (TFA) and glycerol mixed with either 2,4- or 2,5-dihydroxybenzoic acids as two classical UV-MALDI matrices. The highest ion yields of multiply charged peptides were consistently obtained at laser wavelengths for which the chromophore-containing matrix systems exhibited the lowest optical absorption. Best sensitivities were even achieved with the transparent glycerol/TFA mixture and by use of a Nd:YAG-laser operated at 1064nm (or with the OPO laser tuned to a similar wavelength). Experiments with absorbing and transparent sample substrates demonstrated the involvement of substrate absorption. In contrast to the results obtained with the AP ion source, using the same matrix systems in combination with a standard oMALDI2™ ion source, operated at a fine vacuum of ∼1mbar and without an inlet tube, the inverse wavelength dependence was observed. Or in other words, like in standard UV-MALDI under these conditions the highest analyte ion yields were obtained for laser excitation wavelengths corresponding to a high absorptivity of the matrix. Our findings thus point to completely different desorption/ionization processes with the two ion source geometries and laser excitation regimes. We hypothesize that for the AP case the material ablation comprises low-energy laser spallation and that rapid evaporative ionization of small droplets in the heated transfer capillary is responsible for charge production. From an analytical point of view, the production of multiply-charged peptide and protein ions with charge states that for the tested analytes (ranging by mass from 1 to 17kDa) almost fully resembled those generated by nano-electrospray ionization (ESI) could facilitate the coupling of laser-based ion sources with mass analyzers, such as orthogonal-extracting time-of-flight (QTOF) instruments or orbitraps which exhibit only limited m/z ranges.